The present invention, in some embodiments thereof, relates to bacterial resistant plants and methods of generating same.
Ralstonia solanacearum (Rs), a widely distributed, Gram-negative, soil-borne pathogen belonging to the β-subdivision of Proteobacteria, causes a lethal wilting disease of more than 200 plant species including economically important crops such as tomato, potato and banana. The bacterium enters plant roots through wounds, invades the xylem vessels and spreads rapidly to aerial parts of the plant through the vascular system. Rapidly, populations of more than 1010 cells per cm of stem are found. The main virulence factor of Rs is exopolysaccharide (EPS), a long (more than 106 Da) sugar polymer that clogs the xylem and causes wilting symptoms and eventually plant death.
Rs displays a remarkable ability for protein secretion of more than 100 proteins. For example, the Type II secretion system (T2SS) secretes factors including the plant cell wall-degrading pectinases, endo-glucanases, and later, the virulence EPS (FIGS. 1A-C). Type III secretion system (T3SS) secretes infection-promoting effector proteins (T3Es) into host cells to optimize the host environment and suppress plant defense responses following invasion (FIGS. 1A-1C).
The Type III secretion system (T3SS) is a sophisticated molecular machinery of Gram-negative bacteria used to ‘inject’ (translocate) bacterial proteins (effectors) into eukaryotic cells. For this, the T3SS has to assemble into a multi-protein complex, which is comprised of distinct parts; a basal body spanning the two bacterial membranes connected with a cytoplasmic bulb, an attached needle structure resembling a molecular syringe (injectisome/pilus), and a distal needle tip structure that reorganizes into a ‘translocon’, which is a protein complex that inserts into the host cellular membrane. The pilus is built from only one protein subunit. Multiple subunits oligomerize into the pilus structure. This needle structure allows bacterial proteins to be transported through the inner channel, the conduit, of the needle on their way to the host cell (FIGS. 1A-1C).
Thus, the major extracellular component of the T3SS is the needle that extends from the outer-membrane portion of the apparatus and through which runs a 25-Å channel forming the secretion conduit (the helical parameters of the needle are 5.5 subunits per turn; 4.6-Å axial rise per subunit). The needle is formed by a helical assembly of multiple copies (on the order of 100-150) of a single, small (9 kDa) protein. Though there is little homology between the primary sequence of the pilus building blocks of most of the Gram negative bacteria, it is believed that most share some structural homology. In plant pathogenic bacteria, T3SSs are encoded by hrp (hypersensitive response and pathogenicity) genes, which are so named because they are required for bacteria to cause disease in susceptible plants and to elicit the hypersensitive response in resistant plants. Hrp genes were found in almost all major gram-negative bacterial plant pathogens (e.g. Pseudomonas syringae, Xanthomonas spp., Ralstonia solanacearum, and Erwinia spp.), illustrating a central role of the T3SS in mediating diverse plant-bacteria interactions. Thus, typically, the T3SS extracellular pilus is assembled through the stepwise polymerization of a major component (e.g. HrpY in R. solanacearum, HrpA in P. syringae and E. amylovora, HrpE in Xanthomonas campestris, MxiH in Shigella, PrgI in Salmonella and YscF in Yersinia).
As mentioned, although the primary function of type III effectors is to promote plant susceptibility, some effectors are recognized by plant resistance proteins which trigger defense responses, including the hypersensitive response. One method proposed to overcome plant lethal infection by gram-negative bacteria comprises enhancing plant immunity against such pathogens.
U.S. Patent Application Publication No. 20090258825 (He et al.) discloses compositions and methods for enhancing plant defenses against pathogens (e.g. bacterial pathogens). According to their teachings, enhancing plant immunity against the Pseudomonas syringae virulence protein HopM1 is effected by increasing the activity of an ATMIN associated plant protection protein, such as ATMIN7.
U.S. Patent Application Publication No. 20090044296 (Beer et al.) discloses methods of increasing plant growth or imparting disease resistance in plants by the use of nucleic acid molecules configured to increase or decrease expression of a nucleic acid molecule that encodes a HrpN-interacting protein (e.g. HIPM). Deletion analysis disclosed therein showed that the 198-aa N-terminal region of HrpN (harpin) of Erwinia amylovora, the first cell-free elicitor of the hypersensitive response which plays a critical role in the virulence of this pathogen, is required for interaction with HIPM.
Moreover, bacterial wilt is difficult to control because of the soil borne nature of its causal organism. In plants infected by Rs, disease development depends on the action of the Type II and Type III protein secretion systems and mutations in one of these systems leads to non-pathogenic bacteria [Poueymiro et al., Curr. Opin. Microbiol. (2009) 12:44-52].
Roine et al. [Roine et al., FEBS Letters (1997) 417(2): 168-172] showed that once purified, HrpA, the structural protein of Pseudomonas syringae pv. tomato DC3000 pili, alone is sufficient for formation of filament structures undergoing self-assembly.
Taira et al. [Taira et al., Mol Microbiol. (1999) 34(4):737-44] generated insertion mutations in the hrpA gene (e.g. pentapeptide insertions) and created mutated bacteria expressing same. According to their teachings, the carboxy-terminus region of hrpA is crucial for pilus assembly and for bacterial interaction with the affected plant. Moreover, Wei et al. [Wei et al., PNAS (2000) 97(5):2247-2252] identified three single amino acid mutations at the HrpA carboxyl terminus which affect the secretion or regulatory function of the HrpA protein. These results demonstrated an essential role of the Hrp pilus structural gene in protein secretion and coordinate regulation of the type III secretion system in Pseudomonas syringae. Furthermore, Lee et al. [Lee et al., J. Bio. Chem. (2005) 280: 21409-17] disclosed that several pentapeptide-induced nonfunctional HrpA proteins, when expressed in bacteria, exert a strong dominant-negative effect on the function of the wild-type HrpA protein blocking the ability of Pseudomonas syringae to elicit host responses and cause a disease in-vivo. The dominant-negative HrpA mutants were also able to interfere with the self-assembly of wild-type HrpA into pilus in vitro.
Weber at al. [Weber and Koebnik, J. Bacteriology (2005) 187(17): 6175-6186] described hydrophobicity plot analyses of several Hrp pilin proteins, such as HrpE and HrpA from Xanthomonas campestris pv. vesicatoria and HrpY from R. solanacearum, and revealed a common domain organization. These findings suggest that plant-pathogenic bacteria, challenged with the task of overcoming the barrier of a plant cell wall, independently evolved structurally similar proteins. Weber et al. further disclose that pentapeptide insertion mutants in the C-terminal region of HrpE inhibit Hrp pilus assembly in X. campestris pv. Vesicatoria. Morphology studies revealed insertion mutants with shortened Hrp pili. This dominant-negative effect suggests that the mutant variant may interfere with the assembly of the Hrp pilus. U.S. Patent Application Publication No. 20100249234 (Yang et al.) discloses methods of reducing virulence in a bacterium, such as a HrpX/HrpY-type system or a T3SS-type system. The method comprises contacting the bacterium with an effective amount of a phenylpropanoid-type inhibitory compound.
U.S. Patent Application Publication No. 20100099674 (Elofsson et al.) discloses methods for decreasing bacterial virulence in a plant by inhibition of the Type III secretion system using an N-substituted 7-quinolylmethyl amine, in particular one that is further substituted in 5- and 8-position on the quinoline ring.
Additional background art includes U.S. Patent Application Publication No. 20050076406.